In the Hungarian patent specification No. 178,703 (see U.S. Pat. No. 4,294,825) a process has been described for the preparation of a selective anorexogenic substance, i.e. of a product selectively inhibiting the food intake. This substance, which had not been known so far and operated by a new mode of action was prepared from the human plasma and called satietin. It was a partially purified product by far exceeding, however, the activity of similarly acting agents known so far.
The product was purified by ultrafiltration of the plasma and by using a two-step chromatography on Sephadex G-15 and Bio-Gel P-2 gels.
The peptide nature of the substance was determined as well as the quantitative relations of the aminoacids. In addition to the aminoacids, sugar constituents were also found after hydrolysis; thus, this substance acting specifically on the satiety centre was thought to be a glycoprotein.
It had been found by developing the invention that the active fraction could further be resolved, i.e. purified by an additional process. According to the Hungarian patent application No. 2783/81 as modified on 03/03/1982 (see U.S. Pat. No. 4,430,264), a product was obtained which was 3 to 4 times more effective and the chemical composition of which significantly differed from that of the active substance obtained by the process mentioned above. This product could be considered as a chemically pure, uniform, homogeneous active substance. Its physical characteristics and ultimate composition were also determined.
According to the above-mentioned process, the plasma was subjected to an important operation after ultrafiltration; namely, the plasma filtrate was treated with trichloroacetic acid to remove the present serum proteins (albumin, etc.), a defined part of which could pass the Amicon membrane. After the treatment with trichloroacetic acid, the precipitated serum proteins were settled by centrifuging and the pure supernatant containing the biologically effective satietin fractions were further purified on gel columns. In the first step, an ammonium acetate buffer was used for elution from a Sephadex G-15 column, the fractions containing the activity were collected at the void volume and desalted on a Bio-Gel P-2 column after an appropriate concentration. The salt-free fractions could be considered as a substance of high purity, thus, this product was quite useful for the biological study involving all experiments on food intake.
In this step, only peptides and glycopeptides of lower molecular weight remained which had no influence on the satietin activity and were in turn removed by electrophoresis and affinity chromatography. The pure active substance was obtained by lyophilization after fractionating by affinity chromatography on a Con-A Sepharose column and repeated desalination on a Bio-Gel P-2 column. The substance obtained in this way proved to be a glycoprotein with a low peptide (5 to 25%) and a high carbohydrate (60 to 90%) content. The molecular weight of the active substance was in the range of 50,000 to 70,000 daltons, while its isoelectric point was found to be neutral, namely the pI was 7.0 to 7.1.